Springer protocols feed by Immunology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Chimerism Testing by Quantitative PCR Using Indel Markers
Engraftment monitoring is critical for patients after Hematopoietic Stem Cell Transplantation (HSCT). Complete donor chimerism is the goal; therefore, early detection of rejection and relapse is crucial for guiding the patient post HSCT treatment. Quantitative PCR for chimerism testing has been reported to be highly sensitive. In this chapter we discuss the quantitative PCR (qPCR) method using 34 Indel (Insertion and Deletion) genetic markers spread over 20 different chromosomes. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Next-Generation HLA Sequencing Using the 454 GS FLX System
Next-generation sequencing (NGS) of HLA class I and II loci (HLA-A, HLA-B, HLA-C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPB1) is described here in detail using the 454 Life Sciences GS FLX System and Titanium chemistry. An overview of the protocol with our experience on sequence performance efficiencies, read depth and ambiguity analyses using the GS FLX System are also presented. A total of 14 HLA primer pairs with multiplex identifiers (MIDs) are used in clonal, amplicon-based pyrosequencing of up to 44 samples per plate using the GS FLX. Genotype assignment and ambiguity reduction analysis is performed using Conexio ...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Thoracic Organ Transplantation: Laboratory Methods
Although great progress has been achieved in thoracic organ transplantation through the development of effective immunosuppression, there is still significant risk of rejection during the early post-transplant period, creating a need for routine monitoring for both acute antibody and cellular mediated rejection. The currently available multiplexed, microbead assays utilizing solubilized HLA antigens afford the capability of sensitive detection and identification of HLA and non-HLA specific antibodies. These assays are being used to assess the relative strength of donor specific antibodies; to permit performance of virtual ...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Transplantation of the Sensitized Patient: Histocompatibility Testing
A component necessary for successful transplantation of the sensitized patient is timely and high quality support from the histocompatibility laboratory that helps guide selection of the best route to transplantation and the clinical care of the patient. Responsibilities of the laboratory include risk assessment, HLA typing, and accurate antibody characterization. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Composite Tissue Transplantation
Composite tissue transplantation is an emerging new era in transplant medicine and has become a viable reconstructive option for patients with large and devastating tissue defects. Advances in microsurgical techniques, transplant immunology and the development of potent immunosuppressive agents have enabled the realization of such types of transplants. Over the past decade, a rapidly growing number of face and upper extremity transplantations have been performed worldwide with highly encouraging outcomes. However, despite the fact that surgical, immunological and functional results are highly encouraging, the need for long...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Transplantation Tolerance
Tolerance has been defined as graft-specific survival in the absence of continued immunosuppression. The mechanisms of central and peripheral tolerance are discussed in this review, as well as the barriers and limitations in achieving graft-specific tolerance. The need remains for definitive laboratory assays to determine the presence of a tolerant state. Genetic biomarker analysis pre-transplant may allow for better donor: recipient matching, lessening the need for immunosuppression, while post-transplant analysis of biomarkers, certain cytokines, and regulatory leukocytes may permit minimally invasive assessment of graft...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Cell Mediated Rejection
Rejection is the major barrier to successful transplantation and usually results from the integration of multiple mechanisms. Activation of elements of the innate immune system, triggered as a consequence of tissue injury sustained during cell isolation or organ retrieval as well as ischemia–reperfusion, will initiate and amplify the adaptive response. For cell mediated rejection, T cells require multiple signals for activation, the minimum being two signals; antigen recognition and costimulation. The majority of B cells require help from T cells to initiate alloantibody production. Antibodies reactive to donor HLA m...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Antibodies in Transplantation: The Effects of HLA and Non-HLA Antibody Binding and Mechanisms of Injury
Until recently, allograft rejection was thought to be mediated primarily by alloreactive T cells. Consequently, immunosuppressive approaches focused on inhibition of T cell activation. While short-term graft survival has significantly improved and rejection rates have dropped, acute rejection has not been eliminated and chronic rejection remains the major threat to long-term graft survival. Increased attention to humoral immunity in experimental systems and in the clinic has revealed that donor specific antibodies (DSA) can mediate and promote acute and chronic rejection. Herein, we detail the effects of alloantibody, part...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Alloreactivity
The alloimmune response between individuals genetically disparate for antigens encoded within the major histocompatibility complex (MHC) remains a substantial barrier to transplantation of solid organs, tissues, and hematopoietic stem cells. Alloreactivity has been an immunological paradox because of its apparent contradiction to the requirement of MHC restriction for the induction of normal T lymphocyte mediated immune responses. Through crystallographic analyses and experimental systems utilizing murine CD8+ cytolytic T cell clones, major advances have been achieved in understanding the molecular and structural basis of ...
Source: Springer protocols feed by Immunology - January 1, 2013 Category: Allergy & Immunology Source Type: news
Assembly of Matched Alpha/Beta Subunits to HLA Class II Peptide Receptors
Human antigen presenting cells express three human leukocyte antigen (HLA) class II isotypes (DR, DP, and DQ), which are composed of polymorphic α and β subunits. The combination of polymorphic α- and β-chains results in cis (encoded on the same chromosome) or trans (encoded on different chromosomes) combinations. Since some of the α–β combinations may yield mismatched non-functional α–β heterodimers, it is not entirely clear which type of HLA class II peptide receptors are found on the cell surface of antigen presenting cells. We have developed a combination of bioch...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Pulse–Chase Analysis for Studies of MHC Class II Biosynthesis, Maturation, and Peptide Loading
Pulse–chase analysis is a commonly used technique for studying the synthesis, processing and transport of proteins. Cultured cells expressing proteins of interest are allowed to take up radioactively labeled amino acids for a brief interval (“pulse”), during which all newly synthesized proteins incorporate the label. The cells are then returned to nonradioactive culture medium for various times (“chase”), during which proteins may undergo conformational changes, trafficking, or degradation. Proteins of interest are isolated (usually by immunoprecipitation) and resolved by sodium dodecyl sulfat...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Gene Transduction in Human Monocyte-Derived Dendritic Cells Using Lentiviral Vectors
Monocyte-derived dendritic cells (MDDCs) are widely used in the field of human immunology. Although a variety of gene delivery procedures have been used in MDDC, it has remained difficult to achieve robust gene transductions. In this chapter, we describe a procedure for high efficiency gene transduction in human MDDCs using lentiviral vectors. Gene transduction based on HIV-1-derived lentiviral vectors is restricted at the level of reverse transcription by the cellular protein SAMHD1 in MDDCs. Co-transduction of the MDDCs with helper particles derived from SIVmac that contain the viral protein Vpx removes this restriction,...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Analysis of Proteasome Generated Antigenic Peptides by Mass Spectrometry
Mass spectrometry (MS) is today one of the most important analytical techniques in biosciences. The development of electro spray ionization (ESI) as a gentle ionization method, in which molecules are not destroyed, has revolutionized the analytic of peptides. MS is an ideal technique for detection and analysis of peptides generated by in vitro experiments using purified 20S proteasomes. It also provides a convenient and sensitive way to monitor the processing activity of enzymes. The combination of high performance liquid chromatography (HPLC) with ESI-MS allows the analysis of complex samples with separation in their spec...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Preparing Antigens Suitable for Cross-presentation Assays In Vitro and In Vivo
Cross-presentation is defined as the ability of certain professional antigen-presenting cells to take up, process and present extracellular antigens on major histocompatibility class I (MHC-I) molecules to CD8+ T cells. The stimulation of naive cytotoxic CD8+ T cells by this process, termed cross-priming, is involved in many different responses, including those to tumors, pathogens, graft tissues, and self-antigens. Dendritic cells (DCs), a heterogeneous cell population, are endowed with the highest cross-priming capacity. Investigation of their cross-presentation capacities, important both for vaccination and for the indu...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
Isolation of a Specialized, Antigen-Loaded Early Endosomal Subpopulation by Flow Cytometry
Isolation and characterization of antigen-containing endosomes remains difficult utilizing standard purification techniques. Here, we describe a method, which allows isolation of antigen-loaded endosomes, that is based on flow cytometrical analysis and sorting. We specifically isolated antigen-containing endosomes from cells that had taken up fluorochrome-labeled ovalbumin via mannose receptor-mediated endocytosis. The protocol described here allows for the isolation of pure fractions of ovalbumin-containing endosomes and the extraction of proteins from these endosomes for analysis by western blot. Importantly, this protoc...
Source: Springer protocols feed by Immunology - December 3, 2012 Category: Allergy & Immunology Source Type: news
